1. General
As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a fungal pathogen” includes a plurality of fungal pathogens, including mixtures thereof.
As used herein the term “derived from” shall be taken to indicate that a specified integer are obtained from a particular source albeit not necessarily directly from that source.
A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
The embodiments of the invention described herein with respect to any single embodiment shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombining DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated herein by reference:    1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135151;    4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;    5. Perbal, B., A Practical Guide to Molecular Cloning (1984);    6. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Miiler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, 30 Thieme, Stuttgart.    7. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications) Bibliographic details of the publications referred to in this specification are collected at the end of the description
2. Background art
Diseases of the musculoskeletal system such as rheumatoid arthritis (RA), osteoarthritis (OA), disc degeneration (DD), and osteoporosis (OP) are a major cause of morbidity throughout the world. These diseases have a substantial influence on health and quality of life and inflict an enormous cost on health systems.
The aetiology of OA is considered to be multi-factorial with ageing, mechanical, hormonal and genetic factors all contributing to varying degrees. OA emerges as a clinical syndrome when these etiological determinants result in sufficient joint tissue damage to cause synovial inflammation and the appearance of the symptoms of pain and impairment of function.
RA is thought to arise as a consequence of extrinsic and/or intrinsic triggering of an autoimmune response in genetically susceptible individuals. The aggressive inflammation initiated in the joints of RA patients by the activation of their immune system is manifest by the release of pro-inflammatory cytokines, proteinases and free radicals. All of these mediators have the ability to promote the destruction of cartilage, bone and other intra-articular joint tissues leading to further impairment of joint function and progression of the disease. While both OA and RA show common pathological features of cartilage destruction and synovial inflammation, the origins and temporal history of these events are clearly distinct. Nevertheless, destruction of joint cartilage is common to both OA and RA and there is now strong evidence that the breakdown and release of the matrix components from cartilage play a significant role in the chronicity of these diseases.
Cartilage may be considered as an anisotropic biomaterial composed essentially of a three-dimensional fibrous network of type II collagen fibrils copolymerised with types IX and XI collagens embedded in a proteoglycan (PG) rich hydrated extracellular matrix. Type II collagen accounts for over 90% of the total collagen of adult cartilage while the type IX content is only 1-2%.
Type IX collagen is a heteropolymer consisting of three genetically distinct alpha chains, α1(IX), α2 (IX) and α3 (IX) whose molecular structure and amino acid sequences have been described (Pihlajamaa T, et al. Characterisation of recombinant human type IX collagen, association of α chains into homotrimeric and heterotrimeric molecules. J Biol Chem, 274: 22464-22468, 1999)(U.S. Pat. No. 6,127,523, Oct. 3, 2000). The type IX α-chains contain 3 triple-helical domains, COL1, COL2 and COL3 and four non-collagenous domains, NC1, NC2, NC3 and NC4. The NC1 and NC3 regions contain cysteine residues that can form intramolecular disulfide linkages between the α-chains. Only the α1(IX) chain contains a NC4 domain (see FIG. 1) and the sequence from chick sternia has been shown to consist of 243 amino acid residues with a calculated molecular mass of 27,139 Da and overall positive charge (Vasios G, Nishimura I, Konomi H, van der Rest M, Cartilage Type IX collagen-proteoglycan contains a large amino-terminal globular domain encoded by multiple exons. J Biol Chem 263: 2324-2329, 1988).
The type IX collagen heteropolymer is linear except that the NC3 domain acts like a hinge allowing the NC4 region of the α1 chain to project away from the other chains. Interestingly, a single chondroitin sulfate chain is attached to the NC3 domain.
Although only a minor component of cartilage, type IX collagen provides an important role in maintaining the type II collagen fibrous network assembly which is essential for the optimum physical function of this weight-bearing tissue. Type IX collagen resides on the surface of the type II collagen fibrils to which it is covalently linked via, at least 2 trivalent pyridinoline cross-links, particularly at sites where the type II fibril network intersects. By cross-linking the type II collagen fibrils type IX collagen would appear to constrain the expansion caused by the imbibition of water molecules attracted into the tissue by the trapped negatively charged proteoglycan (PG) aggregates (referred to herein as aggrecans). In addition the positively charge centres on the globular NC4 domain also provides a potential binding site for the polyanionic aggrecans.
The aggrecans of cartilage are macro-molecular aggregates of PG subunits which are non-covalently attached along the length of a hyaluronic acid chain. Each aggrecan may contain 20-50 PG subunits and their interaction with the HA backbone is stabilised by ternary interactions with link protein. The PG subunits consist of a protein core to which up to 100 GAG chains are covalently linked. The major GAG substituents of the PGs are the chondroitin sulfates (ChS) and keratan sulfate.
In addition to the collagens and proteoglycans, cartilage also contains a large number of non-collagenous proteins, the most abundant being cartilage oligomeric protein (COMP), cartilage matrix protein (CMP), and thrombospondin. COMP is considered to be a key structural component of the cartilage matrix since it interacts with type IX collagen and plays a role in the development and assembly of type II collagen fibrils (Holden P, et al. Cartilage oligomericmatrix protein interacts with type IX collagen and disruptions to these interactions identify a pathogenic mechanism in a bone displasia familly. J Biol Chem, 276: 6046-6055, 2001). In the early stages of arthritis when cartilage breakdown is increased, PGs, type IX collagen and COMP fragments are some of the first matrix components to be released into synovial fluid by the action of endogenous proteinases. These products of cartilage breakdown have been shown to be antigenic and can induce an inflammatory response within arthritic joints thereby contributing to the rate of disease progression.
In arthritic diseases, the excessive breakdown of cartilage and bone and the concomitant elicitation of an inflammatory reaction provoked by the release of autoantigens are responsible for their chronicity. However, there is accumulating evidence to indicate that these matrix molecules could also be responsible for the initiation of joint diseases by acting as autoantigens. Indeed, systemic administration of type II collagen or other matrix components together with adjuvants to laboratory animals have been used to produce animal models of arthritic disease (Creamer M A, Rosloniec E F, Kang A H. The cartilage collagens: a review of their structure, organization and role in the pathogenesis of experimental arthritis in animals and human rheumatic disease, J Mol Med, 76: 275-288, 1998).
In the case of cartilage collagens, this knowledge has led to the development of means of treating rheumatic diseases by using the concept of oral tolerisation (Weiner, H L, Komagata Y. Oral tolerance and the treatment of Rheumatoid Arthritis. Seminars Immunopath, 20: 289-308, 1998). Thus it has been shown that suppression of type II collagen-induced arthritis in animal models can be achieved by oral administration of low doses of type II collagen. Oral tolerisation of arthritic patients by administration of type II collagen has also been shown to be effective clinically and therapeutic effects have been reported with small (less than 100 mg) daily doses of type II collagen antigens. Studies using rheumatoid and juvenile rheumatoid arthritis patients confirmed the efficacy and safety of low daily oral doses of type II collagen derived from chick sterna (Trentham D, et al. Effects of oral administration of type II collagen on rheumatoid arthritis. Science, 261: 1727-1730, 1993) (Barnett et al, A pilot study of oral type II collagen in the treatment of juvenile rheumatoid arthritis. Arthritis. Rheum. 39:623-628, 1996). Other studies have indicated that cartilage collagens obtained from bovine sources were less effective than those prepared from chick cartilage.
Unfortunately, some studies have found that high doses of type II collagen may in fact exacerbate disease since, as already indicated, this cartilage derived antigen is also an arthritogen and it is difficult to accurately determine how much of this antigen would be effective or detrimental to the arthritic process. With regard to other cartilage collagens types X, XI are arthritogenic, while type IX collagen has been reported to be not as effective as type II collagen as a tolerant in some animal arthritis models (Lu S et al, Different therapeutic and bystander effects by intranasal administration of homologous type II and type IX collagens on the collagen-induced arthritis and Pristane-induced arthritis in rats. Clin Immunol. 90:119-127, 1999).
In another study using recombinantly produced in-tact native type IX collagen it was found that when it was given orally to B10 congenic mice this collagen was able to ameliorate the arthritis produced by prior inoculation with type II collagen (collagen induced arthritis, CIA). The recombinant type IX collagen was also tested for immunogenicity in other murine models and unlike type II collagen, it failed to induce overt arthritis in mice immunized with this protein (Myers L K et al, Immunogenicity of recombinant Type IX collagen in murine collagen-induced arthritis. Arthritis Rheum. 46:1086-1093, 2002).
The discrepancy between the Lu et al (1999) and Myers et al (2002) may be explained by differences in the purity and molecular size of the type IX collagen preparations. Recombinant technology is considered the only means of obtaining pure native type IX collagen since very little can be extracted from the cartilage matrix without using proteolysis as it is covalently bound to the type II collagen fibrils. The most common proteinase used to extract type IX collagen from cartilages is pepsin which is a commercial preparation generally derived from porcine stomach mucosa. Pepsin is not present in cartilage. This enzyme hydrolyses the non-collagenous domains of the type IX molecule releasing the native COL1, COL2, and COL3 triple helical segments which are isolated from the mixture by precipitation leaving the partially degraded non-helical NC domains in solution.
The endogenous proteinases responsible for the normal turnover of type IX collagen in cartilaginous tissues are presently unknown. However, the matrix metalloproteinase (MMP) family, the serine proteinases and cysteine proteinases are all known to degrade type IX collagen in vitro in both the helical (COL) and non-helical (NC) domains.